Cardiomyocyte-specific Piezo1 deficiency mitigates ischemia-reperfusion injury by preserving mitochondrial homeostasis

Abstract

Ca²⁺ overload and mitochondrial dysfunction play crucial roles in myocardial ischemia-reperfusion (I/R) injury. Piezo1, a mechanosensitive cation channel, is essential for intracellular Ca²⁺ homeostasis. The objective of this research was to explore the effects of Piezo1 on mitochondrial function during myocardial I/R injury. We showed that the expression of myocardial Piezo1 was elevated in the infracted area of I/R and cardiomyocyte-specific Piezo1 deficiency (Piezo1^△Myh6) mice attenuated I/R by decreasing infarct size and cardiac dysfunction. Piezo1^△Myh6 regulated mitochondrial fusion and fission to improve mitochondrial function and decrease inflammation and oxidative stress in vivo and in vitro. Mechanistically, myocardial Piezo1 knockout alleviated intracellular calcium overload to normalize calpain-associated mitochondrial homeostasis. Our findings indicated that Piezo1 depletion in cardiomyocytes partially restored mitochondrial homeostasis during cardiac ischemia/reperfusion (I/R) injury. This study suggests an innovative therapeutic strategy to alleviate cardiac I/R injury.

Keywords: Calpain; Cardiomyocytes; I/R; Inflammation; Mitochondria; Piezo1.

Fig. 1

Ischemia-reperfusion injury induced an upregulation of Piezo1. WT male mice and Piezo1^td/Tdt male mice underwent the I/R injury (30min/4h). NMCMs were extracted from both WT and Piezo1^td/Tdt mice and subjected to 8-h hypoxia and 1-h reoxygenation. (A) Western blot analysis and quantitative data of Piezo1 expression in the infarcted and non-infarcted regions of the hearts. (B) Relative expression levels of mRNA for Piezo1 in the infarcted area or non-infarcted area. (C) Representative immunohistochemical staining images and statistical data of Piezo1 expression in the infarcted and non-infarcted regions of the hearts. Scale bar, 1 mm. Detailed photographs are displayed below. Scale bar, 100 μm. (D) Representative images of Piezo1 stained with antibodies against RFP (red). Scale bar, 30 μm. (E) Western blot analysis and quantitative data of Piezo1 expression in NMCMs in Ctrl or H/R groups. (F) Relative expression levels of mRNA for Piezo1 in NMCMs in Ctrl or H/R groups. (G) Representative images of Piezo1 in NMCMs from Piezo1^td/Tdt mice. Scale bar, 25 μm. n = 4 for Western blot analysis, n = 6 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 2

Myocardial Piezo1 knockout alleviated cardiac I/R injury. (A) Representative Evans Blue & TTC staining images of infarct size (IF) and area at risk (AAR) in hearts from Piezo1^fl/fl and Piezo1^△Myh6 mice subjected to I/R(30min/24h) or sham. Scale bar, 1 mm. (B–C) Representative images ang statistical data of TUNEL staining in Piezo1^fl/fl and Piezo1^△Myh6 mice with sham or I/R (30min/24h). Scale bar, 25 μm. (D) Serum LDH concentrations in each group of mice. (E) Representative images and statistical data (G) for live (green) dead (red) staining. Scale bar, 75 μm. (F) Representative images and statistical data (H) of TUNEL positive cells in NMCMs. Scale bar, 75 μm (I) LDH levels in NMCMs from each group. (J) NMCMs viability assayed by CCK8. (K–L) Cardiac gross morphology and the ratio of heart weight to body weight in mice suffering from heart failure. Scale bar, 2 mm. (M–Q) Representative M-mode echocardiograms (M), statistical data of EF, FS, LVIDd and LVIDs (N–Q) for Mice suffering from heart failure. n = 5 for each group. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 3

Myocardial Piezo1 knockout reduced I/R-induced cardiac inflammation. (A) Representative H&E staining images of heart. Scale bar, 100 μm. Detailed photographs are displayed below. Scale bar, 20 μm. (B–E) Representative immunofluorescence staining images and statistical data of the MPO positive cells (B&E), F4/80 positive cells (C&D). Scale bar, 25 μm. (F–G) Representative dot plots and statistical data from flow cytometry analysis for assessing leukocyte percentage (FVD⁻CD45⁺) and neutrophils percentage (FVD⁻CD45⁺CD11b⁺Ly6G⁺). (H) Relative expression levels of IL1β, IL6, TNF-α, MCP-1, MMP9 mRNA of hearts. The heatmap displays the logarithmic values of gene expression levels. (I–L) Relative expression levels of mRNA for IL1β, IL6, TNF-α, MCP-1 of NMCMs. n = 4 for flow cytometry results, n = 6 for heatmap, and n = 5 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 4

Piezo1 deficiency balanced mitochondrial dynamics in H/R treated NMCMs. (A) Representative immunofluorescence staining images and statistical data of mitochondrial lentgh. Scale bar, 25 μm. Detailed photographs are displayed below. Scale bar, 5 μm. (B) The co-localization and statistical data of Drp1 and mitochondria. Scale bar, 25 μm. Detailed photographs are displayed below. Scale bar, 5 μm. (C) Western blot analysis and quantitative data of Drp1, p-Drp1(S616), p-Drp1(S637), Fis1, Mff, OPA1, Mfn1 and Mfn2 in NMCMs. (D) Relative expression levels of mRNA for Drp1, Mff, Fis1, Mfn1, Mfn2 and OPA1 of hearts. The heatmap displays the logarithmic values of mRNA expression. n = 4 for Western blot analysis, n = 6 for heatmap, and n = 5 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 5

Piezo1 deletion inhibits mitochondrial fission, to protect NMCMs from reoxygenation-induced dysfunction. (A–C) Representative images of cellular ROS and mitochondrial ROS along with their quantitative data. Scale bar, 100 μm. (D–E) Representative images and statistical data of MMP in NMCMs. Scale bar, 75 μm. (F–G) Western blot analysis and statistical data of the protein expression of Mn-SOD in NMCMs. (H) Quantitative analysis of ATP levels of NMCMs. n = 4 for Western blot analysis, n = 5 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 6

Mitochondrial oxidative stress inhibition mitigates inflammation. (A) Representative images of DHE positive cells. Scale bar, 25 μm. (B) Representative H&E staining images of hearts. Scale bar, 100 μm. Detailed photographs are displayed below. Scale bar, 20 μm. (C) Relative expression levels of IL1β, IL6, TNF-α, MCP-1 mRNA of hearts. The heatmap displays the logarithmic values of gene expression levels. (D) Representative images of cellular ROS and mitochondrial ROS in NMCMs. Scale bar, 100 μm. n = 5 for each group.

Fig. 7

Myocardial mitochondrial dynamics balance is regulated by Piezo1/Ca²⁺/Calpain axis. (A–C) Representative photographs and statistical data of [Ca²⁺]_i and [Ca²⁺]_m. Scale bars, 25 μm. (D) Calpain activity in heart tissues in each group of mice. (E) Measurements of calpain activity in each group of NMCMs. (F) Measurement of calpain activity in NMCMs treated with H/R with or without PD150606. (G) Representative immunofluorescence staining images of mitochondrial length of NMCMs treated with H/R with or without PD150606. Scale bar, 10 μm. Detailed photographs are displayed below. Scale bar, 2 μm. (H) Western blot analysis of Drp1, p-Drp1 (S616), p-Drp1 (S637), OPA1, Mfn1 and Mfn2 expression in NMCMs. (I) The co-localization of Drp1 and mitochondria. (J) Representative TUNEL staining images of NMCMs. Scale bar, 75 μm. (K) Representative photographs for live (green) dead (red) staining. Scale bar, 75 μm. n = 4 for Western blot analysis, and n = 5 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 8

Tamoxifen-induced Piezo1 depletion in myocardial cells alleviated I/R injury. (A) Representative Evans Blue & TTC staining images of hearts from Piezo1^△MCM and Piezo1^fl/fl mice subjected to I/R (30min/24h) or sham. Scale bar, 1 mm. (B–C) Representative photomicrographs and quantitative data of TUNEL positive cells in Piezo1^fl/fl and Piezo1^△MCM mice with sham or I/R (30min/24h). Scale bar, 25 μm. (D) Serum LDH concentrations in mice. (E) Representative H&E staining photographs of hearts. Scale bar, 100 μm. Enlarged images are shown at the bottom. Scale bar, 20 μm. (F–I) Representative immunofluorescence staining images of the MPO positive cells and F4/80 positive cells along with their quantitative data. Scale bar, 25 μm. (J) Relative mRNA expression levels of IL1β, IL6, TNF-α, MCP-1, MMP9 in hearts. The heatmap displays the logarithmic values of gene expression levels. n = 6 for heatmap, and n = 5 for other results. Data are presented as mean ± SEM. ∗P < 0.05.

Fig. 9

Graphic illustration on Piezo1 Activation Under I/R Promotes Mitochondrial Fission, Oxidative Stress, and Inflammation via Calpain, Exacerbating I/R Injury.